Fish & Shellfish Immunology 32 (2012) 598e608

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Fish & Shellfish Immunology

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Short communication Molecular characterization of three L-type lectin from channel catfish, Ictalurus punctatus and their responses to Edwardsiella ictaluri challenge

Hao Zhang a,b, Eric Peatman a, Hong Liu a, Tingting Feng a, Liqiao Chen b, Zhanjiang Liu a,* a The Fish Molecular Genetics and Biotechnology Laboratory, Department of Fisheries and Allied Aquacultures and Program of Cell and Molecular Biosciences, Aquatic Genomics Unit, Auburn University, 203 Swingle Hall, Auburn, AL 36849, USA b School of Life Science, East China Normal University, Shanghai 200062, China article info abstract

Article history: L-type lectins have a leguminous lectin domain and can bind to high-mannose type oligosaccharides. In Received 22 November 2011 the secretory pathway, L-type lectins play crucial roles in selective trafficking, sorting and tar- Received in revised form geting. Three L-type lectins were cloned in the channel catfish, Ictalurus punctatus, the 53 kDa endo- 22 December 2011 plasmic reticulum ER-Golgi intermediate compartment protein (ERGIC-53), the vesicular integral protein Accepted 23 December 2011 of 36 kDa (VIP36) and VIP36-like. Phylogenetic analysis indicated that the catfish genes are orthologous Available online 6 January 2012 to their counterparts in other species. Southern blot analysis demonstrated that all three L-type lectin genes are likely single-copy genes in the catfish genome. Analysis of expression in healthy tissues using Keywords: Lectin quantitative real time RT-PCR indicated that all three genes are expressed widely in all tested tissues, but Immune response with strong tissue preference of expression: ERGIC-53 was found to be abundantly expressed in the liver, Catfish VIP36 was found to be abundantly expressed in the head-kidney, whereas VIP36-like was found to be ERGIC-53 abundantly expressed in the brain. Upon infection with Edwardsiella ictaluri, expressions of the three VIP36 genes all had significant up-regulation in the head-kidney, but had distinct expression patterns: ERGIC- 53 was gradually induced with the highest expression 7 days after challenge in the head-kidney, but was down-regulated in the liver, spleen, and brain. VIP36 was highly induced in the head-kidney, and 3 days after challenge in the brain, but was not up-regulated in any other tissues or timepoints after challenge. Expression levels of the catfish VIP36-like appeared to also respond to infection, albeit with differing patterns among the tested tissues. Taken together, our results indicate that all three L-type lectin genes may be involved in the immune responses of catfish after infection with E. ictaluri. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction Drosophila [2].InCaenorhabditis elegans, ERGIC-53 can affect the transport of yet other glycoproteins [2]. Recent studies have shown L-type lectins, including the 53 kDa that in human ERGIC-53 results in combined deficiency of (ER)-Golgi intermediate compartment protein ERGIC-53 [1] and factor V and factor VIII, causing an autosomal recessive bleeding VIP36 [2], are intracellular lectins that are located in luminal disorder characterized by coordinated reduction of both clotting compartments of the secretary pathway and function in the traf- [4,6,7]. ficking, sorting and targeting of maturing glycoproteins [3]. ERGIC- The 36 kDa vesicular integral protein (VIP36) is a type I 53 genes in many species are annotated as lectin, mannose-binding membrane glycoprotein with a CRD similar to that of ERGIC-53, and 1 (LMAN1) while VIP36 genes are annotated as lectin, mannose- which is thought to act as a cargo receptor for glycoprotein quality binding 2 (LMAN2). ERGIC-53 is a mannose-specific lectin that control in the Golgi [8,9]. VIP36 has specificity for high-mannose cycles between the ER and the ER-Golgi intermediate compartment type glycans of the Man6e9 but its efficient binding requires the (ERGIC) [4]. It has been shown that ERGIC-53 is a cargo transport additional presence of an a-substituted asparagine residue [2,5], receptor for some glycoproteins in ER export [2,5]. ERGIC-53 may and the binding of VIP36 to high-mannose type glycans is inde- þ affect b-integrin traffic, as demonstrated by a mutant of ERGIC-53 pendent of Ca2 [2,10]. Knock-down of VIP36-like mRNA using that can cause a b-integrin-related developmental defect in siRNA in HeLa cells suggested that VIP36 may also function as an ER export receptor [11].

* Corresponding author. Tel.: þ1 334 844 8727; fax: þ1 334 844 9208. It is now apparent that ERGIC-53 and VIP36 share similar L-type E-mail address: [email protected] (Z. Liu). lectin domains and mannose-binding selectivity but have distinct

1050-4648/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2011.12.009 H. Zhang et al. / Fish & Shellfish Immunology 32 (2012) 598e608 599 functions within the early secretory pathway [5]. Selective protein [32]. The signal peptides and conserved domains were searched by transport and sorting in the secretory pathway is fundamentally SMART program (http://smart.embl-heidelberg.de/). important for both healthy and diseased organisms. Within plant innate immune systems L-type lectins have been hypothesized to play a role as pattern recognition receptors while recent work in 2.2. Phylogenetic analysis mammalian systems indicates potential roles in infectious responses and phagocytosis [12e14]. Amino acid sequences of the LMAN, ERGIC-53 and VIP36 genes Although sequences of L-type lectins from teleost fish can be from various species were downloaded from NCBI and Ensembl identified in public gene databases, they have not been character- Genome Browser. A phylogenetic tree was constructed using the ized previously or examined for potential roles in immune neighbor-joining method within the Molecular Evolutionary responses. In channel catfish (Ictalurus punctatus), the primary Genetics Analysis (MEGA 4) package [33]. aquaculture species in the United States [15], a large number of genes involved in innate immunity have been characterized including antimicrobial peptides [16e18], Toll-like receptors [19], 2.3. Tissue sampling and RNA extraction NOD-like receptors [20], and a large number of chemokines, cyto- kines, and acute phase response proteins [21e25]. Additionally, we To determine in various healthy tissues, have previously characterized several other groups of lectins from samples of 11 tissues including brain, gill, heart, head-kidney, catfish including a C-type mannose-binding lectin [26]. However, trunk-kidney, intestine, liver, muscle, skin, spleen, and stomach no information is known about the nature of the L-type lectins and were collected. Due to small sample size of each individual fish, we their relationship with the immune response. Catfish production collected samples into three pools with tissues from 15 fish in each suffers heavy losses due to enteric septicemia of catfish (ESC), pool. The tissues were flash-frozen in liquid nitrogen and stored in caused by a Gram-negative intracellular bacterium Edwardsiella a 80 C ultrafreezer until RNA preparation. Tissues were homog- ictaluri [27]. ESC in its acute form is characterized by gastroenteric enized under liquid nitrogen using the RNeasy Plus Mini Kit (Qia- septicemia and, under artificial challenge, often results in heavy gen, Valencia, CA, USA) to extract total RNA following the mortalities as early as four days after onset of infection [28,29].To manufacturer’s instructions. First-strand cDNA was synthesized better understand the crucial innate immune response of channel using an iScript cDNA Synthesis Kit (Bio-Rad, USA) following the catfish in the context of ESC, and the possible involvement of L-type manufacturer’s instructions. lectins in the innate immune system of channel catfish, here we characterized three L-type lectins, conducted phylogenetic analysis, and analyzed their expression after ESC challenge. 2.4. Southern blot analysis

2. Materials and methods To determine the genomic copy number of L-type lectin genes in channel catfish, Southern blot analysis was conducted as previously fl m 2.1. Identification and sequencing of catfish L-type lectin cDNAs described [26]. Brie y, 10 g of genomic DNA isolated from each of three individual adult channel catfish was digested with 30 units of Based on the cDNA sequences of L-type lectin from zebrafish, we the restriction endonucleases EcoRI,Hind III or Pst I (New England m searched the channel catfish expressed sequence tags (ESTs) data- Biolabs, Beverly, MA) in a 25 L reaction at 37 C. The digested DNA base [30,31] in NCBI using BLAST. The clone CBPN21042 (GenBank samples were electrophoresed on a 0.8% agarose gel. After elec- ID: FD339357.1 and FD339356.1), CBPN25640 (GenBank ID: trophoresis, the gel was submerged in 0.25 N HCl for 15 min, then in FD345714.1 and FD345715.1) and CBPO11108 (GenBank ID: denaturation and neutralization buffer for 30 min, respectively. The FD041401.1 and FD041402.1) were determined to encode the DNA was transferred to an Immobilon positively-charged nylon putative complete cDNAs of catfish ERGIC-53, VIP36, and VIP36-like membrane (Millipore, Bedford, MA) by capillary transfer for 18 h fi genes. These clones were re-sequenced to confirm the sequences using 20 SSC buffer. The DNA was xed to the membrane using using T7 and SP6 primers (Table 1), using the BigDye Terminator a UV cross-linker (Stratagene, La Jolla, CA) with the auto-crosslink v3.1 Cycle Sequencing kit (Applied Biosystems, Foster City, CA, USA) settings. The membrane was hybridized with cDNA probes ampli- fied using primers listed in Table 1. After pre-hybridization for 2 h with salmon sperm DNA and hybridization with a 32P-dCTP labeled Table 1 probe at 63 C for 16 h, the membrane was washed twice with wash Primers used in this study. buffer 1 (2 SSC, 0.1% SDS) and one time with wash buffer 2 (0.5 Primer name Primer sequence 50-30 Application SSC, 0.1% SDS), and then exposed to a piece of X-ray film overnight T7 TAATACGACTCACTATAGGG For sequencing at 80 C. SP6 ATTTAGGTGACACTATAG

18SrRNA-F GAGAAACGGCTACCACATCC For quantitative real time 18SrRNA-R GATACGCTCATTCCGATTACAG RT-PCR 2.5. Bacterial challenge and sample collection ERGIC-531F CGAGCACATCATCCAGAGAA ERGIC-53 1R CTGCTTCCTTTTGGCTTTTG Tissues were collected from 15 healthy fish immediately before VIP361F GTAGCAAGCAAGGCTCCATC VIP361R ACAGCCAGGCCTACAAAATG the challenge experiments began. Bacterial challenge was con- VIP36-like1F ATATCGACGGTCAGCACGA ducted as previously described [34], using E. ictaluri as the path- VIP36-like1R CCGGAGTACTGTCAGCTGGT ogen, and samples collected at 4 h, 24 h, 3 d and 7 d after challenge.

ERGIC-53e2F CCAAAGAGGAGCAGGACAAG For generation of Southern At each time point, head-kidney, trunk-kidney, intestine, liver and ERGIC-53e2R CGTCTGAACAACGACGAAGA blot probes spleen tissues were collected from 45 fish in three pools (15 fish VIP36e2F GTAGCAAGCAAGGCTCCATC each pool) of both the challenge group and the control group. Then VIP36e2R CAGACTGACACTGGGCTCAA RNA was isolated and cDNA synthesized as described in Section 2.3. VIP36-likee2F CCTCAAACACGACACCTTCC The cDNA samples were subsequently used for determination of VIP36-likee2R CCAGTGGCTGTAAACCACAA gene expression by quantitative real time RT-PCR. 600 H. Zhang et al. / Fish & Shellfish Immunology 32 (2012) 598e608

Fig. 1. The cDNA and amino acid sequences of L-type lectins ERGIC-53 (A), VIP36 (B) and VIP36-like (C). Based on the cDNA sequences of L-type lectin from zebrafish, the clone CBPN21042 (GenBank ID: FD339357.1 and FD339356.1), CBPN25640 (GenBank ID: FD345714.1 and FD345715.1) and CBPO11108 (GenBank ID: FD041401.1 and FD041402.1) were determined to encode the putative complete cDNAs of catfish ERGIC-53, VIP36, and VIP36-like genes using BLAST against the channel catfish expressed sequence tags (ESTs) database in NCBI. These clones were re-sequenced to confirm the sequences using T7 and SP6 primers. For all three L-type lectins, the cDNA sequences are presented along with the deduced amino acid sequences with the putative signal peptide sequences being underlined with solid lines, the L-type lectin superfamily domains being underlined with dotted lines, and the C-terminal transmembrane domains being boxed in the rectangle box. H. Zhang et al. / Fish & Shellfish Immunology 32 (2012) 598e608 601

Fig. 1. (continued).

2.6. Quantitative real time RT-PCR analysis different tissues using analysis of variance (ANOVA) (SPSS 14.0 package, SPSS Inc., New York, USA). Differences were considered First-strand cDNA was synthesized using an iScript cDNA significant at p < 0.05. Synthesis Kit (Bio-Rad, USA). Quantitative real time RT-PCR was used to examine the mRNA expressions of three genes in different 3. Results and discussion tissues and following bacterial challenge. The reactions were per- formed on a Bio-Rad CFX96 (Bio-Rad, USA), using the SsoFastÔ 3.1. Identification and sequencing of catfish L-type lectin cDNAs Ò EvaGreen Supermix kit (Bio-Rad, USA). Total cDNA (225 ng) was used in each reaction. The 18S rRNA gene was used as an internal The channel catfish ERGIC-53 cDNA contains an open reading control for normalization of expression levels [26,34]. The primers frame (ORF) of 1506 bp encoding 502 amino acids with a 427 bp 30- used in quantitative real time RT-PCR are listed in Table 1. The cycle untranslated region (UTR). The sequence of the VIP36 cDNA time (Ct) values were compared and converted to fold differences consists of 30-UTR of 707 bp with a poly (A) tail and an ORF of by the relative quantification method using the Relative Expression 1020 bp encoding a polypeptide of 348 amino acids. The VIP36-like Software Tool 384 v.1 (REST) [35]. Comparisons were made in the cDNA contains an ORF of 1506 bp encoding 340 amino acids and 602 H. Zhang et al. / Fish & Shellfish Immunology 32 (2012) 598e608

Fig. 1. (continued). a1200bp30-UTR with a typical polyadenylation signal sequence binding site is generally localized toward the apex of this dome AATAAA. The ERGIC-53, VIP36 and VIP36-like protein possess [12]. In the sugar binding mechanisms of most L-type lectin putative signal sequences of 30, 35, and 21 amino acids in length, domains, metal ions are required for ligand binding [9,12,36]. respectively (Fig. 1A for ERGIC-53, Fig. 1B for VIP36, Fig. 1C for BLASTP searches indicated that catfish ERGIC-53 and VIP36 are VIP36-like). highly similar to these genes from other species. The L-type lectin The L-type lectin superfamily domain is composed of b-sheets superfamily domain (including the carbohydrate-binding site and connected by short loops and b-bends formed a dome-shaped beta- the metal binding site) was found in ERGIC-53 (Arg38-Glu263), barrel carbohydrate recognition domain and the carbohydrate- VIP36 (Glu43-Val270) and VIP36-like (Glu29-Val267) (Fig. 2A for H. Zhang et al. / Fish & Shellfish Immunology 32 (2012) 598e608 603

Fig. 2. Multiple sequence alignment of the deduced amino acid sequences of L-type lectins ERGIC-53 (A), VIP36 (B) and VIP36-like (C). The conserved and identical residues are represented by black shading, and conservative substitutions are represented by gray shading. The L-type lectin superfamily domains were underlined; triangles indicate 604 H. Zhang et al. / Fish & Shellfish Immunology 32 (2012) 598e608

Fig. 2. (continued).

ERGIC-53, Fig. 2B for VIP36, Fig. 2C for VIP36-like). C-terminal (55%e65%) between the protein sequences of teleost LMAN2-like transmembrane domains were predicted at residues Leu468- a genes and LMAN2-like b genes suggested a duplication event in Gln480, Val315-Phe337, and Ile306-Tyr328 for ERGIC-53, VIP36, most fish. In catfish, only one gene (LMAN2-like a) was found, and VIP36-like, respectively. In the C-terminus of ERGIC-53 protein, suggesting that either the LMAN2-like b gene has not been iden- there is a double phenylalanine signal and double lysine signal tified from existing genome and transcriptome sequences or that domain (AKKFF) which was found to be necessary for the transport channel catfish may have lost LMAN2-like b during evolution. of glycoproteins between the endoplasmic reticulum and Golgi [1]. Analysis of the deduced amino acid sequences by multiple 3.3. Determination of genomic copy number of L-type lectin sequence alignment indicated that the L-type lectin genes are highly conserved through evolution (Fig. 2A for ERGIC-53, Fig. 2B Southern blot analysis was conducted to determine the copy for VIP36, Fig. 2C for VIP36-like). The catfish L-type lectins have number of the ERGIC-53, VIP36 and VIP36-like genes in the channel 78e90% identity with zebrafish L-type lectin protein sequences and catfish genome. As shown in Fig. 4A, three bands were observed 50e70% identity with other vertebrate L-type lectin protein with EcoRI digest and two bands were observed with Hind III or Pst I sequences such as that of rat, human, frog and bird. digest, indicative of potentially more than a single copy of the ERGIC-53 gene within the catfish genome. However, an examina- 3.2. Phylogenetic analysis of mannose-binding lectin genes tion of restriction sites within the genomic sequence of ERGIC-53 indicated that there were two restriction sites for EcoR I and Hind A phylogenetic tree was constructed using amino acid III and one restriction site for Pst I, confirming a single gene in the sequences of 22 protein sequences. As shown in Fig. 3, channel catfish genome. Similarly, at least two bands were observed with catfish L-type lectin genes fell into three distinct clades: one clade VIP36 gene (Fig. 4B). Examination of the restriction sites within the included all ERGIC-53 (LMAN1) sequences from teleost species and gene sequences revealed the presence of one restriction site for other vertebrate species, one clade included all VIP36 genes EcoR I and Pst I and three restriction sites for Hind III. Taken (LMAN2), whereas the third clade included the catfish VIP36-like together, these data indicated the presence of a single-copy gene (LMAN2-like) sequence along with VIP36-like genes from all for VIP36 with the catfish genome as well. For VIP36-like gene, only teleost species. The third clade divided to two branches: one branch one band was observed with Southern blot analysis (Fig. 4C) for included the catfish VIP36-like gene, a part of the teleost VIP36-like EcoR I and Pst I and two bands for Hind III. Considering there is one genes (LMAN2-like a), and all the other vertebrate species VIP36- restriction sites for Hind III and no restriction site for EcoR I and Pst I like genes, while the other branch only included a paralog of the within the genomic sequence of VIP36-like, the result suggested teleost VIP36-like genes (LMAN2-like b). The higher similarity a single-copy gene for VIP36-like within the catfish genome.

carbohydrate-binding site; asterisks indicate metal binding site; C-terminal transmembrane domains have been boxed with a rectangle box; the double phenylalanine signal and double lysine signal domain (AKKFF) of ERGIC-53 protein has been boxed with dotted line (A). The Genbank accession numbers of genes involved are as below, ERGIC-53 Danio reio, XP_688661; LMAN2 Danio reio, CAN88460; VIP36-like Danio reio, NP_991288; VIP36 Osmerus mordax, ACO09232; ERGIC-53 Homo sapiens, NP_005561; VIP36 Homo sapiens, NP_006807; VIP36-like Homo sapiens, NP_001135764; ERGIC-53 Mus musculus, AAH57165; LMAN2 Mus musculus, AAH55327; VIP36-like Mus musculus, NP_001013392; LMAN2-like Xenopus tropicalis, NP_001136372; ERGIC-53 Gallus gallus, NP_001026570. Fig. 3. Phylogenetic analysis of known L-type lectins with representatives from mammals, birds, frog, and fish. The Genbank accession numL-type lectinbers of genes involved are as below, ERGIC-53 Danio reio, XP_688661; LMAN2 Danio reio, CAN88460; VIP36-like Danio reio, NP_991288; LMAN2-like b Danio reio, NP_001070201; VIP36-like Esox lucius, ACO13933; VIP36 Osmerus mordax, ACO09232; ERGIC-53 Homo sapiens, NP_005561; VIP36 Homo sapiens, NP_006807; VIP36-like 1 Homo sapiens, NP_001135764; VIP36-like 2 Homo sapiens, NP_110432; LMAN1 Mus musculus, AAH57165; LMAN2 Mus musculus, AAH55327; VIP36-like Mus musculus, NP_001013392; LMAN1 Xenopus laevis, NP_001080950; LMAN2 Xenopus tropicalis, NP_001072196; LMAN2-like Xenopus tropicalis, NP_001136372; LOC734646 Xenopus laevis, NP_001089589; ERGIC-53 Gallus gallus, NP_001026570; LMAN1 Taeniopygia guttata, XP_002188765; LMAN2 variant1 Taeniopygia guttata, ACH44464; LMAN2 variant2 Taeniopygia guttata, ACH44465; LMAN2-like Taeniopygia guttata, XP_002193623. And the protein ID of genes involved from Ensembl Genome Browser are as below, LMAN1 Tetraodon nigroviridis, ENSTNIP00000010335; LMAN2 Tetraodon nigroviridis, ENSTNIP00000009077; LMAN2-like a Tetraodon nigroviridis, ENSTNIP00000003410; LMAN2-like b Tetraodon nigroviridis, ENSTNIP00000022042; LMAN1 Takifugu rubripes, ENSTRUP00000031419; LMAN2 Takifugu rubripes, ENSTRUP00000002773; LMAN2-like a Takifugu rubripes, ENSTRUP00000047826; LMAN2-like b Takifugu rubripes, ENSTRUP00000042548; LMAN1 Oryzias latipes, ENSORLP00000007624; LMAN2 Oryzias latipes, ENSORLP00000014117; LMAN2-like a Oryzias latipes, ENSORLP00000009101; LMAN2-like b Oryzias latipes, ENSORLP00000020395; LMAN1 Gasterosteus aculeatus, ENSGACP00000021709; LMAN2 Gasterosteus aculeatus, ENSGACP00000022210; LMAN2-like a Gasterosteus aculeatus, ENSGACP00000022138; LMAN2-like b Gasterosteus aculeatus, ENSGACP00000007115. The topological stability of the neighbor-joining tree was evaluated by 10,000 bootstrapping replications, and the bootstrapping percentage values are indicated by numbers at the nodes. 606 H. Zhang et al. / Fish & Shellfish Immunology 32 (2012) 598e608

Fig. 4. Southern blot analysis of catfish L-type lectin genes ERGIC-53 (A), VIP36 (B), and VIP36-like (C). Genomic DNA of three individual fish was used as marked and digested with EcoRI,Hind III, and Pst I. Molecular weight is shown on the left margin of the gel.

Therefore, all three L-type lectin genes are likely single-copy genes in catfish. This result is consistent with the copy number of these genes among the sequenced teleost genomes such as zebrafish, medaka, stickleback, and Tetraodon, where a single copy was identified from the genome sequences by searching through Ensembl Genome Browser (http://uswest.ensembl.org/index.html).

Fig. 5. Analysis of gene expression of L-type lectins ERGIC-53 (A), VIP36 (B) and VIP36- 3.4. Catfish L-type lectin gene expression in healthy and infected like (C) in various tissues of healthy catfish using quantitative real time RT-PCR. Gene tissues expression levels in different tissues are relative to that in the brain. The 18S rRNA gene was used as an internal control. Comparisons were made in the different tissues using analysis of variance (ANOVA) (SPSS 14.0 package, SPSS Inc., New York, USA). Significant Quantitative real time RT-PCR was used to determine tissue statistical differences (p < 0.05) are indicated by an asterisk. distribution of expression of the three L-type lectin genes in healthy H. Zhang et al. / Fish & Shellfish Immunology 32 (2012) 598e608 607

channel catfish. As shown in Fig. 6, catfish L-type lectin genes were widely expressed in all tested tissues (Fig. 5A for ERGIC-53, Fig. 5B for VIP36, Fig. 5C for VIP36-like) but with distinct patterns of expression. The catfish ERGIC-53 gene had the highest expression in the liver; catfish VIP36 was most abundantly expressed in the head-kidney; and catfish VIP36-like had the highest expression in the brain (p < 0.05). ERGIC-53 was reported as an excellent marker for the ER-Golgi intermediate compartment (ERGIC) and is expressed in all cells of multicellular organisms [1,2,37]. Its L-type lectin domain can carry a segment of about 200 amino acids of glycoprotein from the ER to the Golgi and can recycle between ERGIC and ER [38e40]. As the homolog of ERGIC-53, VIP36 is suggested to have a function in quality control of glycosylation in the Golgi [2]. Due to the lack of a double lysine signal domain, VIP36 recycles between ERGIC and the Golgi, not between the ERGIC and the ER [41e43]. While the roles of L-type lectins are well established in protein transport during homeostatic conditions, their roles in infectious responses have only recently been revealed. Shirakabe et al. [14] demonstrated that VIP36 regulates phagocytosis in macrophages through shedding. Additionally, study of mice with differing susceptibilities to Staphylococcus aureus identified ERGIC-53 (LMAN1) as an important expression and QTL candi- date [13]. In order to determine if catfish L-type lectins are similarly involved in responses to disease infection with the Gram-negative intracellular bacterium E. ictaluri, quantitative real time RT-PCR analysis was conducted to determine the expression patterns of the L-type lectins in infected liver, spleen, head-kidney, and brain. As shown in Fig. 6, the expression of the catfish ERGIC-53 was down-regulated in liver, spleen and brain, respectively, at different time points following infection (Fig. 6A). However, significant up- regulation of the catfish ERGIC-53 gene expression was observed in the head-kidney at 24 h and 7 d (p < 0.05) (Fig. 6A). The expression of catfish VIP36 was induced in head-kidney at 24 h, 3 d, 7 d, and in brain by 3 d (p < 0.05) (Fig. 6B). The transcript of VIP36-like gene was significantly up-regulated in liver at 7 d, in spleen at 4 h, and in head-kidney at 24 h (p < 0.05) (Fig. 6C); however, down-regulation of VIP36-like was observed at 3 d and 7 d in the spleen and at 3 d in the head-kidney (p < 0.05). Head- kidney might be the most active tissue for the expressions of three L-type lectin genes during the immunity-stimulation. In particular, ERGIC-53 was only up-regulated in head-kidney. Additionally, L- type lectins such as VIP36 may be mediating macrophage-derived responses to infection in the head-kidney and sites of entry (brain) through a putative role in phagocytosis. VIP36-like may have the widest expressions in liver, spleen and head-kidney, but only the response times were different. The expression of VIP36-like was firstly up-regulated in spleen, and then the increased protein concentration may inhibit the further expression of VIP36-like gene in spleen. The same pattern of VIP36-like expression also appeared in head-kidney. The acute bacterial infection of E. ictaluri likely impacts protein trafficking, leading to differential expression of the catfish L-type lectins. Future work is needed to more clearly elucidate the implicated cell types and functional roles of L-type lectins during disease.

Fig. 6. Analysis of expression of catfish L-type lectin genes ERGIC-53 (A), VIP36 (B), and VIP36-like (C) using quantitative real time RT-PCR in the liver, spleen, head-kidney, Acknowledgments and brain tissues after infection with Edwardsiella ictaluri at various time points (0 h, 4 h, 24 h, 3 d and 7 d). Fold change is expressed as the ratio of gene expression after E. ictaluri challenge to the control group at same time point as normalized with 18S This project was supported by grants from the USDA AFRI rRNA gene. Error bars indicate standard error and asterisks indicate statistical signif- Animal Genome Basic Genome Reagents and Tools Program (USDA/ < icance (p 0.05). NRICGP award# 2009-35205-05101 and 2010-65205-20356). Hao Zhang was supported by a scholarship from the China Scholarship Council (CSC). 608 H. Zhang et al. / Fish & Shellfish Immunology 32 (2012) 598e608

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